Cloning and sequence determination of a complementary DNA related to human liver microsomal cytochrome P-450 S-mephenytoin 4-hydroxylase

Abstract

A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.

Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future

“…An enzyme that was active in the metabolism of mephenytoin was purified in the laboratories of both Fred Guengerichs and Meyers. Antibodies against the purified enzymes were used to screen human lgt11 cDNA libraries and CYP2C8 and CYP2C9 were cloned [40]. The expressed enzymes were not found to hydroxylate mephenytoin but rather tolbutamide [41].…”

Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

“…It is found rather exclusively in family II of the P450 gene superfamily which is involved in the metabolism of foreign compounds such as drugs (Figure 6). Except for a few isoenzymes belonging to subfamily IIC, where Ser is substituted by Thr (which can also serve as phosphoryl group acceptor) all sequences of family II isoenzymes contain this sequence (Khani et al, 1987;Umbenhauer et al, 1987). Arginine residue 125 of the region, which is also part of the kinase recognition sequence and resides in the close vicinity of the phosphorylated serine residue, is highly conserved in P450 isoenzymes.…”

Fluorescencein situ hybridization analysis of chromosomal localization of three human cytochrome P450 2C genes (CYP2C8, 2C9, and 2C10) at 10q24.1

“…Shimada et aL (1986) first isolated two similar forms of human liver P450 enzymes responsible for (S)-mephenytoin 4-hydroxylation, termed P-450~.~_ 1 and P-450~p_~. Furthermore, Umbenhauer et al (1987) and Ged et al (1988) have isolated four cDNA clones that contained nearly full-length DNA sequences, from a bacteriophage Xgtl 1 library prepared from a single human liver using anti-P450~e_l as a probe, and designated them as MP-12 (CYP2C8), MP-20 (CYP2C8), MP-4 (CYP2C9), and MP-8 (CYP2C10). Recently, CYP2C9 and CYP2C10 enzymes have been reported to catalyze tolbutamide methyl hydroxylation and hexobarbital 3'-hydroxylation (Brian et al, 1989;Srivastava et al, I991 ;Yasumori et aL, 1991), and CYP2C8 enzyme is found to be one of the benzo-(a)pyrene 3-hydroxylases (Yun et al, 1992).…”